The recovery, structure, and function of human blood leukocytes after freeze-preservation

Crowley, James P.; René, Anthony A.; Valeri, C. R.

doi:10.1016/0011-2240(74)90106-0

Cited by 35 publications

(13 citation statements)

References 19 publications

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“…2, P > 0.5, ANOVA) indicating functional integrity of PBMC after thawing. These data are consistent with previous findings that cryopreservation did not affect integrins on mononuclear cells (8,11).Overall, based on this limited set of markers, there was no indication for diminution or selection by cryopreservation of PBMC subpopulations, thus confirming and extending previous reports (2,4,12). In addition, our finding that cryopreservation of PBMC from several volunteers apparently did not affect their capacity to interact with activated endothelial cells now lends experimental evidence to the notion that cryopreservation is a useful tool to preserve native human immune cells for long-term or repeated functional analyses, at least regarding dynamic interactions with endothelial cells.…”

supporting

confidence: 92%

Phenotypic and functional traits of peripheral blood mononuclear cells retained by controlled cryopreservation: implications for reliable sequential studies of dynamic interactions with endothelial cells

Lockmann

Schön

2013

Experimental Dermatology

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Many immunological experiments would be greatly facilitated if peripheral blood mononuclear cells (PBMC) preserved important functional properties over longer periods of time. Regarding adhesive interactions with endothelial cells, a crucial step of inflammatory processes, this had not been investigated yet. We demonstrate that PBMC subsets subjected to controlled cryopreservation retain their phenotypic traits inasmuch as the proportion of viable T cells, monocytes/macrophages and B cells was comparable with their freshly isolated counterparts. More importantly, we demonstrate for the first time that the procedure does not impede crucial adhesion-mediated dynamic interactions with endothelial cells. Using a flow chamber system, freshly isolated and cryopreserved PBMC showed similar rolling and firm adhesion on TNF-a-activated endothelial cells under shear flow as compared to freshly isolated PBMC from the same donors. Thus, our observation is an important prerequisite for functional studies of leucocyte recruitment when sequential investigations with material from the same patients are required.Key words: endothelial cell -leukocyte rolling -method for preserving PBMC functions Accepted for publication 20 February 2013Peripheral blood mononuclear cells (PBMC) are indispensable for many immunological analyses, both in basic and clinical research. Long-term or repeated studies require PBMC storage while reliably preserving functional traits. Since the first successful preservation of mouse lymphocytes (1), different freezing protocols have been developed. Phenotypic traits of lymphocytes remained stable after freezing at À80°C, while monocytes, macrophages and granulocytes were more prone to damage (2). Programmed freezing and mechanical freezing methods appeared to be comparable regarding recovery and viability of PBMC (3). Concerning apoptosis, storage of PBMC at À150°C was superior compared to storage at À30°C or to storage of whole-blood samples at either temperature (4,5).Notwithstanding a fair number of approaches to store PBMC, we were surprised to find that virtually all of these earlier studies had a peculiar view on a limited array of cellular functions or mere phenotypic characterization (5-7). Thus, the capability of cryopreserved PMBC to properly interact with endothelial cells, a crucial early step in inflammatory reactions, still had to be established. Using a dynamic flow chamber system, we provide here the first experimental evidence for intact functional traits of PBMC after cryopreservation regarding rolling on and adhesion to cultured endothelial cells.Towards this end and with ethical approval, venous blood (20-40 ml) was collected from a total of six healthy volunteers (five female non-smokers, one male smoker; age range 22-28 years). PBMC were isolated using Ficoll-Paque PLUS density gradient centrifugation and washed with Hank's buffered salt solution (HBSS). PBMC were labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE, 2 lM in HBSS) for flow chamber analysis dir...

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supporting

confidence: 92%

Phenotypic and functional traits of peripheral blood mononuclear cells retained by controlled cryopreservation: implications for reliable sequential studies of dynamic interactions with endothelial cells

Lockmann

Schön

2013

Experimental Dermatology

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“…1,16 Recovery of CD34 þ cells (67.472.0%) compared to CFU-GM (67.371.0%, P40.05) demonstrates that the recovery of CD34 þ cells assayed by 7-AAD accurately reflects the HPC proliferative capacity. Therefore, enumeration of CD34 þ cells and CFU-GM could be used as reliable and reproducible post thaw assays for HPC.…”

Section: Discussionmentioning

confidence: 99%

Effects of incubation temperature and time after thawing on viability assessment of peripheral hematopoietic progenitor cells cryopreserved for transplantation

Yang

Acker

Cabuhat

et al. 2003

Bone Marrow Transplant

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Summary:Three widely used viability assessments were compared: (1) membrane integrity of nucleated cells using trypan blue (TB) exclusion and a fluorometric membrane integrity assay (SYTO 13 and propidium iodide), (2) enumeration of viable CD34 þ cells, and (3) clonogenic assay (granulocyte-macrophage colony-forming units, CFU-GM). Post thaw peripheral hematopoietic progenitor cells (HPC) were incubated at 0, 22, and 371C for 20-min intervals before assessment. The recovery of viable nucleated cells assessed by TB and SYTO/PI decreased significantly with time at incubation temperatures of 22 and 371C (Po0.05), and correlated with the concentration of mononuclear cells (MNC) (r ¼ 0.936, Po0.05). The decrease in recovery of viable nucleated cells was slower when thawed cells were incubated at 01C compared with 221C or 371C. The recovery, measured by absolute viable CD34 þ or CFU-GM, was not affected by 2 h post thaw incubation (P40.05) at 0, 22, and 371C (P40.05). There were no significant differences in the measured recovery of viable CD34 þ cells and CFU-GM at all incubation times (P40.05) and temperatures (P40.05). Both CFU-GM and absolute CD34 þ cells can be used as post thaw viability assays for HPC cryopreserved for transplantation.

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“…However, these studies showed considerable variability (0-304%) after cryopreservation 4 and an increased mean of 1.22% viable CD34 þ cells before cryopreservation to a mean of 2.16% after cryopreservation. 5 Other studies 4,[6][7][8][9][10][11][12][13][14][15][16][17][18][19] have shown more convincing results, but the washing of the postcryopreservation component for the enumeration of CD34 þ cells does not represent the actual number of CD34 þ cells in the DMSO-containing HPC products that are infused in the patients. The above procedures used for CD34 þ cell enumeration and CFU-GM assay (if performed) had not been validated and standardized, and the increased viability after cryopreservation (4100%) made the proposal even more dubious.…”

Section: Cd34mentioning

confidence: 99%

“…9,10 However, the number of nucleated cells, particularly neutrophils, is decreased during cryopreservation and during post-thaw incubation due to cell loss and clumping of the damaged cells. 11,12 In dual-platform CD34 þ enumeration assays, this decrease in the number of nucleated cells results in an apparent increase in the percentage of CD34 þ cells when nucleated cell count is used as the denominator to calculate the total post-cryopreservation CD34 þ cells. Even when using a validated single-platform CD34 þ enumeration technique to obtain absolute CD34 þ cells, assays did not account for cells lost during cryopreservation or post-thaw washing, 13 nor were comparisons made with proliferation of progenitor cells (granulocyte-macrophage colony-forming units (CFU-GM)).…”

Section: Cd34mentioning

confidence: 99%

Association of post-thaw viable CD34+ cells and CFU-GM with time to hematopoietic engraftment

Yang

Acker

Cabuhat

et al. 2005

Bone Marrow Transplant

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Summary:In all, 78 peripheral hematopoietic progenitor cell collections from 52 patients were evaluated using our previously published validated post-thaw assays at the time of collection and following transplantation by assessment of viable CD34 þ cells, and granulocytemacrophage colony-forming units (CFU-GM) cryopreserved in quality control vials. The median (range) post-thaw recovery of viable CD34 þ cells and CFU-GM was 66.4% (36.1-93.6%) and 63.0% (28.6-85.7%), respectively, which did not show significant correlation with the engraftment of either neutrophils (P ¼ 0.136 and 0.417, respectively) or platelets (P ¼ 0.88 and 0.126, respectively). However, the reinfused viable CD34 þ cells/ kg of patient weight pre-or post-cryopreservation showed significant correlation to engraftment of neutrophils (P ¼ 0.0001 and 0.001, respectively) and platelets (P ¼ 0.023 and 0.010, respectively), whereas CFU-GM pre-or post-cryopreservation was significantly correlated to neutrophils (P ¼ 0.011 and 0.007, respectively) but not to platelets (P ¼ 0.112 and 0.100, respectively). The results show that post-cryopreservation assessment of viable CD34 þ cells or CFU-GM is as reliable a predictor of rapid engraftment as that of pre-cryopreservation measures. Therefore, the post-cryopreservation number of viable CD34 þ cells or CFU-GM should be used to eliminate the risks of unforeseen cell loss that could occur during cryopreservation or long-term storage.

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The recovery, structure, and function of human blood leukocytes after freeze-preservation

Cited by 35 publications

References 19 publications

Phenotypic and functional traits of peripheral blood mononuclear cells retained by controlled cryopreservation: implications for reliable sequential studies of dynamic interactions with endothelial cells

Phenotypic and functional traits of peripheral blood mononuclear cells retained by controlled cryopreservation: implications for reliable sequential studies of dynamic interactions with endothelial cells

Effects of incubation temperature and time after thawing on viability assessment of peripheral hematopoietic progenitor cells cryopreserved for transplantation

Association of post-thaw viable CD34+ cells and CFU-GM with time to hematopoietic engraftment

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